Protein delivery by secretory gland expression

ABSTRACT

Secretory gland cells, particularly pancreatic, hepatic, and salivary gland cells, are genetically altered to operatively incorporate a gene which expresses a protein which has a desired therapeutic effect on a mammalian subject. The expressed protein is secreted directly into the bloodstream to obtain therapeutic levels of the protein thereby treating the patient in need of the protein. The transformed secretory gland cells provide long term or short term therapies for diseases associated with a deficiency in a particular protein or which are amenable to treatment by overexpression of a protein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.08/591,197, filed Jan. 16, 1996, now U.S. Pat. No. 5,885,971 which is acontinuation-in-part of U.S. application Ser. No. 08/410,660, filed Mar.24, 1995, now U.S. Pat. No. 5,837,693, each of which are incorporatedherein by reference.

FIELD OF THE INVENTION

This invention relates generally to the field of genetic transformationof cells in vivo, more particularly to in vivo transformation ofsecretory gland cells by introduction of the transforming nucleic acidinto a secretory gland via a secretory gland duct.

BACKGROUND OF THE INVENTION

The ability to replace defective or absent genes has attracted wideattention as a method to treat a variety of human diseases (Crystal 1995Science 270:404), Lever et al. 1995 Gene Therapy. Pearson Professional,New York p. 1-91; Friedmann 1996 Nature Med. 2:144). Although originallyintended as a means of correcting inherited disorders in certainpopulations of somatic cells, gene-based therapy can be a useful meansto supply exogenous gene products to the circulatory system for thetreatment of a wide range of systemic disorders that involvedeficiencies in circulating proteins, such as hormones, growth factors,and clotting proteins (Lever et al. 1995 supra; Buckel 1996 TiPS17:450), as well as a means of administering other polypeptide drugs.The success of this application depends upon developing effectivemethods to both manufacture the desired protein in vivo and then secreteit into blood (Crystal 1995 supra; Lever et al. 1995 supra).

Currently, DNA-based therapy (i.e., gene therapy) is carried out in avariety of ways but involves two general protocols. In the first method,referred to as ex vivo gene therapy, cells are extracted from anindividual and subjected to genetic manipulation. After genetic materialhas been properly inserted into the cells, the cells are implanted backinto the individual from which they were removed. Persistent, in vivoexpression of the newly implanted genetic material after transplantationof the transformed cells has been successful (see Morgan et al., Science237:1476 (1987); and Gerrard et al., Nat. Genet. 3:180 (1993)). In thesecond approach to DNA-based therapy, referred to as in vivo genetherapy, cells within a living organism are transformed in situ withexogenous genetic material.

Several different methods for transforming cells can be used inaccordance with either the ex vivo or in vivo transfection procedures.For example, various mechanical methods can be used to deliver thegenetic material, including the use of fusogenic lipid vesicles(liposomes incorporating cationic lipids such as lipofection; seeFelgner et al., Proc. Nat. Acad. Sci. U.S.A. 84:7413-7417 (1987));direct injection of DNA (Wolff, et al., Science (1990) 247:1465-1468);and pneumatic delivery of DNA-coated gold particles with a devicereferred to as the gene gun (Yang et al., Proc. Natl. Acad. Sci. U.S.A.1990; 87:1568-9572). Morsy et al. reviews several of the differenttechniques useful in transformation of cells ex vivo or in vivo andprovides citations of numerous publications in each area (Morsy et al.,JAMA 270:2338-2345 (1993)).

One method of particular interest for delivery of genetic materialinvolves use of recombinant viruses to infect cells in vivo or ex vivo.In these methods, a virus containing the desired genetic material isallowed to infect target cells within the subject. Upon infection, thevirus injects its genetic material into the target cells. The geneticmaterial is then expressed within the target cell, providing forexpression of the desired genetic material. However, it would bepreferable to avoid introduction of the desired genetic material byviral infection for a number of reasons. For example, viral infectionresults in delivery of viral DNA in addition to the desired geneticmaterial, which may in turn result in undesirable cellular effects suchas, adverse immune reactions, productive viral replication, and adverseintegration events.

There is a need in the field for a method for delivery of geneticmaterial into a cell in vivo to provide for expression of the introducedpolynucleotide and secretion of the gene product it encodes into thebloodstream. The present invention addresses this problem.

SUMMARY OF THE INVENTION

Secretory gland cells are genetically altered to operatively incorporatea gene which is expressed by the genetically altered secretory glandcell to produce a polypeptide which is subsequently secreted into thebloodstream. Specifically, the invention involves introduction of anucleotide of interest into a secretory gland via the duct system (e.g.,by retrograde ductal administration) to transform a secretory glandcell.

In one embodiment the invention features genetic alteration of cells oftwo secretory glands (e.g., the liver and the pancreas).

In another embodiment, the invention features transformation ofpancreatic cells with insulin-encoding nucleic acid to provide forexpression and secretion of insulin at levels sufficient to maintain asubstantially euglycemic state in a subject having a diabetic syndrome.

A primary object is to provide a non-invasive method of protein delivery(i.e., the method involves introduction of the nucleic acid of interestfrom outside the body (i.e., from the duct system of particular glands)wherein cells of a secretory gland, preferably the pancreas, salivarygland, or liver of a mammal are genetically modified to express abiologically active and therapeutically useful polypeptide, whichpolypeptide is secreted into the circulatory system of the individual.

Another object is to produce genetically transformed secretory glandcells which cells have incorporated into their genome genetic materialwhich, when expressed, produces a biologically active andtherapeutically useful protein which is secreted into the circulatorysystem.

An advantage of the present invention is that both long and short termtherapy can be provided for diseases wherein individuals are sufferingfrom the disease due to a deficiency in a particular protein, or bysupplying an exogenous protein having a desired activity (e.g,antimicrobial activity).

These and other objects, advantages and features of the presentinvention will become apparent to those persons skilled in the art uponreading the details of the methodology and compositions as more fullyset forth below.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a map of the pFGH construct, which contains the human growthhormone (hGH) genomic sequence.

FIG. 2 is a map of the pFGH.CMV construct, which contains the hGHgenomic sequence operably linked to the CMV promoter.

FIG. 3 is a map of the pFGH.chymo construct, which contains the hGHgenomic sequence operably linked to the chymotrypsin B promoter.

FIG. 4 is a graph showing the levels of tissue expression of hGHexpression in the pancreas of rats after retrograde injection witheither a control containing no DNA or a test sample containing a hGHconstruct.

FIG. 5 is a graph showing the serum levels of hGH in rats afterretrograde pancreatic injection with either a control containing no DNAor a test sample containing a human growth hormone construct.

FIG. 6 is a graph showing the correlation between pancreatic tissueexpression and serum levels of hGH.

FIG. 7 is a graph showing tissue expression of hGH followingtransformation of salivary gland cells by intraductal injection.

FIG. 8 is a graph showing regulation of plasma levels of hGH expressedfrom transformed salivary gland cells.

FIG. 9 is a graph showing regulation of plasma levels of hGH expressedfrom transformed pancreatic cells.

FIG. 10 is a map of the pBAT16.hInsG1.M2 construct, which contains DNAencoding an altered form of human insulin.

FIG. 11 is a graph showing the glucose response instreptozotocin-treated rats having pancreatic cells transformed witheither human insulin (open bars) or green fluorescent protein (GFP;striped bars).

FIG. 12, is a graph showing the blood glucose levels in control rats(mock-treated; closed squares), streptozotocin-treated rats (opensquares), and streptozotocin rats treated by transformation ofpancreatic cells with DNA encoding human insulin (closed circles).

FIG. 13 is a graph showing expression of hGH in the plasma of controlrats (no DNA) and of rats in which hGH-encoding DNA was introduced intothe liver by intraductal injection.

FIG. 14 is a graph showing the relative amounts of hGH in the pancreatictissue of rats that received either pFGH (control), pFGH.chymo,pFGH.RSV, pFGH.RSV, or pFGH.CMV by intraductal administration to thepancreas.

FIG. 15 is a graph showing the relative amounts of hGH in the pancreatictissue of rats that received either no DNA (mock-transformed), pFGH.CMV,pFGH.CMV premixed with lipofectin, or pFGH.CMV premixed with adenovirus.

FIG. 16 is a graph showing the relative levels of plasma hGH in ratsthat received either pFGH (control), pFGH.chymo, pFGH.RSV, pFGH.RSV, orpFGH.CMV by intraductal administration to the pancreas.

FIG. 17 is a graph showing the relative amounts of plasma hGH in ratsthat received either no DNA (mock-transformed), pFGH.CMV, pFGH.CMVpremixed with lipofectin, or pFGH.CMV premixed with adenovirus byintraductal administration to the pancreas.

FIG. 18 is a graph showing the relative levels of plasma hGH in ratsthat received no DNA (control), received hGH-encoding DNA viaintraductal delivery to the liver, received hGH-encoding DNA viaintraductal delivery to the pancreas, or received hGH-encoding DNA viaintraductal delivery to both the liver and pancreas.

FIG. 19 is a graph showing the relative levels of hGH expression inpancreas tissue following administration of DNA to both pancreas andliver or to pancreas alone. The graph shows tissue levels of hGH afteradministration of a control (no DNA) to both pancreas and liver(left-most bar); administration of pFGH.CMV to both pancreas and liver(center bar); and pFGH.CMV to pancreas alone (right-most bar).Adenovirus was admixed with the construct as an adjuvant.

FIG. 20 is graph showing the relative levels of hGH expression insalivary gland tissue in rats that received either no DNA (controlrats), pFGH.CMV, pFGH.CMV premixed with lipofectin, or pFGH.CMV premixedwith adenovirus.

FIG. 21 is a graph showing stimulation of hGH secretion into the plasmaof rats that received hGH-encoding DNA by intraductal injection intoboth the pancreas and liver.

FIG. 22 is a graph showing the blood glucose levels ofstreptozotocin-treated rats (diabetic) that received either no DNA (opensquares) or received human insulin-encoding DNA by intraductal injectioninto the pancreas (closed squares) over a three day period.

FIG. 23 is a graph showing the plasma insulin levels ofstreptozotocin-treated rats (diabetic) that received either no DNA (opensquares) or received human insulin-encoding DNA by intraductal injectioninto the pancreas (closed squares)over a three day period.

FIG. 24 is a graph showing the blood glucose levels (over a six dayperiod) of streptozotocin-treated rats (diabetic) that received eitherno DNA (open squares) or received human insulin-encoding DNA byintraductal injection into the pancreas (closed squares).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Before the present method of genetically transforming secretory glandcells and methods for protein delivery are described, it is to beunderstood that this invention is not limited to the particularmethodology, protocols, cell lines, secretory glands, vectors andreagents described as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention which will be limited only by theappended claims.

It must be noted that as used herein and in the appended claims, thesingular forms "a", "and", and "the" include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to "asecretory gland cell" includes a plurality of such cells and referenceto "the transformation vector" includes reference to one or moretransformation vectors and equivalents thereof known to those skilled inthe art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention belongs. Although any methods, devicesand materials similar or equivalent to those described herein can beused in the practice or testing of the invention, the preferred methods,devices and materials are now described.

All publications mentioned herein are incorporated herein by referencefor the purpose of describing and disclosing the cell lines, vectors,and methodologies which are described in the publications which might beused in connection with the presently described invention. Thepublications discussed herein are provided solely for their disclosureprior to the filing date of the present application. Nothing herein isto be construed as an admission that the invention is not entitled toantedate such a disclosure by virtue of prior invention.

Definitions

By "secretory gland" is meant an aggregation of cells specialized tosecrete or excrete materials not related to their ordinary metabolicneeds. Secretory glands include salivary glands, pancreas, mammaryglands, thyroid gland, thymus gland, pituitary gland, liver, and otherglands well known in the art.

By "exocrine gland" is meant a ducted gland or portion of a ducted glandthat releases its products externally relative to the body, e.g., eitherinto the internal cavities such as the ocular and nasal cavities, thelumen of the gastrointestinal tract, or onto the surface of the body.

By "salivary gland" is meant a gland of the oral cavity which secretessaliva, including the glandulae salivariae majores of the oral cavity(the parotid, sublingual, and submandibular glands) and the glandulaesalivariae minores of the tongue, lips, cheeks, and palate (labial,buccal, molar, palatine, lingual, and anterior lingual glands).

By "pancreas" is meant a large, elongated, racemose gland situatedtransversely behind the stomach, between the spleen and the duodenum.The pancreas is composed of an endocrine portion (the pars endocrina)and an exocrine portion (the pars exocrina). The pars endocrina, whichcontains the islets of Langerhans, produces and secretes proteins,including insulin, directly into the bloodstream. The pars exocrinacontains secretory units and produces and secretes a pancreatic juice,which contains enzymes essential to protein digestion, into theduodenum.

By "retrograde ductal injection" is meant the administration of a liquidor other material into the fluid contents of the duct system of anexocrine gland in a direction opposite to the normal flow of that fluid,either at the external orifice of the duct system or through its wall."Retrograde ductal injection" can be a single, discontinuousadministration or continuous administration (i.e., perfusion).

By "transformation" is meant a genetic change induced in a cellfollowing incorporation of new DNA (i.e., DNA exogenous to the cell).Where the cell is a mammalian cell, the genetic change may be achievedby introduction of the DNA into the genome of the cell.

By "transfection" is meant the transformation of a cell with DNA from avirus.

By "transformed cell" is meant a cell into which (or, where theintroduced DNA is incorporated into the genome, into an ancestor ofwhich) has been introduced, by means of recombinant DNA techniques, aDNA molecule encoding a protein of interest.

By "nucleic acid of interest" is meant any DNA or RNA molecule whichencodes a polypeptide or other molecule which is desirable foradministration to a mammalian subject for expression of the productencoded by the nucleic acid of interest and delivery of the encodedproduct into the blood stream of the mammalian subject. The nucleic acidis generally operatively linked to other sequences which are needed forits expression such as a promoter. The term "DNA of interest" is used asshorthand herein to refer to the nucleic acid of interest.

By "construct" is meant a nucleic acid molecule which contains thenucleic acid of interest (e.g., the DNA of interest), generally operablylinked to a promoter for expression of the polypeptide encoded by thenucleic acid of interest. "Constructs" as used herein is generally meantto refer a nucleic acid molecule that facilitates expression of apolypeptide encoded by the nucleic acid to be introduced into asecretory gland cell.

By "vector" is meant any compound, biological or chemical, thatfacilitates transformation of a secretory gland cell with a DNA ofinterest. Exemplary biological vectors include viruses, particularlyattenuated and/or replication-deficient viruses. Exemplary chemicalvectors include lipid complexes and naked DNA constructs.

By "naked DNA" or "naked nucleic acid" or DNA sequence and the like ismeant a nucleic acid molecule that is not contained within a viralparticle, bacterial cell or other encapsulating means that facilitatesdelivery of nucleic acid into the cytoplasm of the target cell. Nakednucleic acid can be associated with means for facilitating delivery ofthe nucleic acid to the site of the target cell (e.g., means thatfacilitate travel into the target cell of the nucleic acid through thealimentary canal, protect the nucleic acid from stomach acid, and/orserve to penetrate intestinal mucus) and/or to the surface of the targetepithelial cell.

By "promoter" is meant a minimal DNA sequence sufficient to directtranscription. "Promoter" is also meant to encompass those promoterelements sufficient for promoter-dependent gene expression controllablefor cell-type specific, tissue-specific or inducible by external signalsor agents; such elements may be located in the 5' or 3' regions of thenative gene.

By "secretory gland specific promoter" is meant a promoter which directsexpression of an operably linked DNA sequence when bound bytranscriptional activator proteins, or other regulators oftranscription, which are unique to a specific type of secretory glandcell. For example, by "salivary gland specific promoter" is meant asecretory gland specific promoter which directs expression in a salivarygland cell. A salivary amylase promoter is an example of a salivarygland specific promoter. By "pancreas specific promoter" is meant asecretory gland specific promoter which directs expression in apancreatic cell. Examples of pancreas specific promoters include apancreatic amylase promoter and an insulin promoter.

By "operably linked" is meant that a DNA sequence and a regulatorysequence(s) are connected in such a way as to permit gene expressionwhen the appropriate molecules (e.g., transcriptional activatorproteins) are bound to the regulatory sequence(s).

By "operatively inserted" is meant that the DNA of interest introducedinto the cell is positioned adjacent a DNA sequence which directstranscription and translation of the introduced DNA (i.e., facilitatesthe production of, e.g., a polypeptide encoded by a DNA of interest).

By "mammalian subject" or "mammalian patient" is meant any mammal towhich intravenous protein delivery is desired, including human, bovine,equine, canine, and feline subjects.

By "euglycemia" or "euglycemic state" is meant a state associated with alevel of blood glucose that is normal or nearly normal, particularlyrelative to the levels of blood glucose in a subject having a disease orcondition associated with hyperglycemia. In humans, euglycemiacorrelates with blood glucose levels in the range of 70 mg/dl to 130mg/dl.

The terms "synergistic," "synergistic effect," and the like are usedherein to describe improved effects (e.g, an increase in tissueexpression levels in one or more secretory glands, an increasedresponsiveness to hormonal stimulation to elicit secretion of apolypeptide of interest, or a decrease in an undesirable phenotype) bycombining one or more aspects of the invention (e.g., by transformationof more than one secretory gland in a single subject, or bytransformation of a secretory gland(s) with multiple constructs encodingthe same or different polypeptides).

Overview of the Invention

The present invention features methods for genetically altering asecretory gland cell (i.e., secretory gland cell transformation) andmethods of delivering a protein using the methods of geneticallyaltering secretory gland cells. More specifically the invention featuresmethods for delivery of a protein or other product encoded by a nucleicacid sequence of interest to a mammalian subject by expression of a DNAof interest in cells within a secretory gland of a mammalian patient(i.e., by in vivo gene therapy). Preferably, the transformed secretorygland cells expressing the protein encoded by the DNA of interestsecrete a therapeutically effective amount of the protein into thebloodstream of the mammalian patient. Preferably, the secretory glandinto which the DNA of interest is introduced and expressed is thepancreas, a salivary gland, or the liver. In short, the inventionfeatures a delivery system that involves introduction of a nucleic acidsequence encoding a product of interest (e.g., a protein) into asecretory gland cell (e.g., a salivary gland cell, hepatocyte, orpancreatic cell, particularly exocrine cells of salivary gland, liver,or pancreas), expression of the encoded protein, and delivery of theprotein into the blood stream by secretion of the protein by thetransformed secretory gland cell.

The present invention preferably uses either naked DNA or DNA premixedwith adjuvants (e.g., lipofectin or viral particles). It is notnecessary to incorporate the DNA into viral particles in order toachieve transformation of secretory gland cells and provide expressionof the polypeptide of interest at physiologic/therapeutic levels in thebloodstream.

An important feature of the invention is the use of exocrine cells ofglands of the gastrointestinal tract (i.e., pancreas, liver, salivarygland) to produce and secrete therapeutic proteins into blood. While itis well understood that exocrine cells secrete into the lumen of theglands' ducts (i.e. in an exocrine direction), with the exception of theliver (i.e., the hepatocytes secrete cellular products in bothdirections, e.g. blood proteins into blood and bile satls into theintestinal lumen), it is not widely appreciated that exocrine cells canalso secrete significant amounts of protein into the systemiccirculation. For example, exocrine proteins such as α-amylase (salivaryglands), pepsinogen (gastric glands), various digestive enzymes from theexocrine pancreas, salivary gland kallikreins and nerve growth factor(Liebow, 1988 Pancreas 3:343-351) are normal constituents of blood. Inthe pancreas, substantial quantities of digestive enzymes are releasedinto the circulation (Saito et al., 1973 Jpn. J. Physiol. 23:477-95;Isenman et al. 1997 Proc. Natl. Acad. Sci (USA) 74:4068-4072; Papp etal. 1980 Acta Physiol. Acad. Sci. Hung. 56:401-410; Geokas et al., 1980Am. J. Physiol. 238:238-246; Miyasaka et al. 1981 Am. J. Physiol.241:170-175; Grendell et al. 1982 Am. J. Physiol. 243:54-59). Endocrinesecretion can be greatly enhanced by common secretory stimulants (Saitoet al., supra; Isenman et al. supra; Miyasaka et al. supra; Grendell etal. supra). As much as 20-25% of the total secreted product can bereleased into blood as a consequence of stimulation (Grendell et al.supra). The present invention takes advantage of the discovery thatexocrine gland cells can be transformed with a desired DNA sequence andsecrete the encoded polypeptide into the bloodstream rather than only orprimarily into the gastrointestinal tract.

In addition to the advantages described above, the invention alsopermits access to the cells of secretory glands without invasiveprocedures. For example, it is possible to cannulate either thecollecting duct of a major salivary gland through its orifice in themouth, or the common bile or pancreatic duct by means of endoscopicretrograde cholangiopancreatography (ERCP). These are common diagnosticprocedures performed on awake patients. The non-invasive methods of theinvention allow delivery of the DNA of interest in a safe manner thatsubstantially avoids the inflammatory and immunological responsesassociated with other means of DNA delivery.

The invention also takes advantage of the protein-producing capacity ofsecretory gland cells. This advantage is particularly useful for theproduction of hormones such as hGH and insulin, which have shorthalf-lives in blood and are cleared quickly. The cells of the exocrineglands are the body's major protein synthesizing and secreting systems.For example, the human exocrine pancreas manufactures and secretesapproximately 20 g of protein daily. According to the present invention,even a small proportion of protein synthesized by secretory glandsprovides enough secreted product to provide therapeutic protein levelsfor the treatment of most diseases of circulating proteins.

The invention will now be described in further detail.

Constructs

Any nucleic acid construct having a eukaryotic promoter operably linkedto a DNA of interest can be used in the invention. The constructscontaining the DNA sequence (or the corresponding RNA sequence) whichmay be used in accordance with the invention may be any eukaryoticexpression construct containing the DNA or the RNA sequence of interest.For example, a plasmid or viral construct (e.g. adenovirus) can becleaved to provide linear DNA having ligatable termini. These terminiare bound to exogenous DNA having complementary, like ligatable terminito provide a biologically functional recombinant DNA molecule having anintact replicon and a desired phenotypic property. Preferably theconstruct is capable of replication in both eukaryotic and prokaryotichosts, which constructs are known in the art and are commerciallyavailable.

The exogenous (i.e., donor) DNA used in the invention is obtained fromsuitable cells, and the constructs prepared using techniques well knownin the art. Likewise, techniques for obtaining expression of exogenousDNA or RNA sequences in a genetically altered host cell are known in theart (see, for example, Kormal et al., Proc. Natl. Acad. Sci. USA,84:2150-2154, 1987; Sambrook et al. Molecular Cloning: a LaboratoryManual, 2nd Ed., 1989, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.; each of which are hereby incorporated by reference withrespect to methods and compositions for eukaryotic expression of a DNAof interest).

Preferably, the DNA construct contains a promoter to facilitateexpression of the DNA of interest within a secretory gland cell.Preferably the promoter is a strong, eukaryotic promoter such as apromoter from cytomegalovirus (CMV), mouse mammary tumor virus (MMTV),Rous sarcoma virus (RSV), or adenovirus. More specifically, exemplarypromoters include the promoter from the immediate early gene of humanCMV (Boshart et al., Cell 41:521-530, 1985) and the promoter from thelong terminal repeat (LTR) of RSV (Gorman et al., Proc. Natl. Acad. Sci.USA 79:6777-6781, 1982). Of these two promoters, the CMV promoter ispreferred as it provides for higher levels of expression than the RSVpromoter.

Alternatively, the promoter used may be a tissue-specific promoter. Forexample, where the secretory gland is the pancreas, the promoter used inthe vector is preferably a pancreas specific promoter, e.g., an insulinpromoter or a pancreas α-amylase promoter; where the secretory gland isa salivary gland, the tissue-specific promoter may be a salivaryα-amylase promoter or mumps viral gene promoter. Both pancreatic andsalivary α-amylase genes have been identified and characterized in bothmice and humans (see, for example, Jones et al., Nucleic Acids Res.,17:6613-6623; Pittet et al., J. Mol. Biol., 182:359-365, 1985;Hagenbuchle et al., J. Mol. Biol., 185:285-293, 1985; Schibler et al.,Oxf. Surv. Eukaryot. Genes, 3:210-234, 1986; and Sierra et al., Mol.Cell. Biol., 6:4067-4076, 1986 for murine pancreatic and salivaryα-amylase genes and promoters; Samuelson et al., Nucleic Acids Res.,16:8261-8276, 1988; Groot et al., Genomics, 5:29-42, 1989; and Tomita etal., Gene, 76:11-18, 1989 for human pancreatic and salivary α-amylasegenes and their promoters; Ting et al., Genes Dev. 6:1457-65, 1992 forhuman salivary α-amylase AMY1C promoter sequences).

The constructs of the invention may also include sequences in additionto promoters which enhance secretory gland specific expression. Forexample, where pancreas specific expression of the DNA of interest isdesired, the construct may include a PTF-1 recognition sequence (Cockellet al., Mol. Cell. Biol., 9:2464-2476, 1989). Sequences which enhancesalivary gland specific expression are also well known in the art (see,for example, Robins et al., Genetica 86:191-201, 1992).

Other components such as a marker (e.g., an antibiotic resistance gene(such as an ampicillin resistance gene) or β-galactosidase) to aid inselection of cells containing and/or expressing the construct, an originof replication for stable replication of the construct in a bacterialcell preferably, a high copy number origin of replication), a nuclearlocalization signal, or other elements which facilitate production ofthe DNA construct, the protein encoded thereby, or both.

For eukaryotic expression, the construct should contain at a minimum aeukaryotic promoter operably linked to a DNA of interest, which is inturn operably linked to a polyadenylation sequence. The polyadenylationsignal sequence may be selected from any of a variety of polyadenylationsignal sequences known in the art. Preferably, the polyadenylationsignal sequence is the SV40 early polyadenylation signal sequence. Theconstruct may also include one or more introns, which can increaselevels of expression of the DNA of interest, particularly where the DNAof interest is a cDNA (e.g., contains no introns of thenaturally-occurring sequence). Any of a variety of introns known in theart may be used (e.g, the human β-globin intron, which is inserted inthe construct at a position 5' to the DNA of interest).

The DNA of interest may be inserted into a construct so that thetherapeutic protein is expressed as a fusion protein (e.g., a fusionprotein having β-galactosidase or a portion thereof at the N-terminusand the therapeutic protein at the C-terminal portion). Production of afusion protein can facilitate identification of transformed cellsexpressing the protein (e.g., by enzyme-linked immunosorbent assay(ELISA) using an antibody which binds to the fusion protein).

The Nucleic Acid (DNA) of Interest

The DNA of interest can be any DNA encoding any protein for whichintravenous therapy is desirable. For example, intravenous proteintherapy is appropriate in treating a mammalian subject having aninherited or acquired disease associated with a specific proteindeficiency (e.g., diabetes, hemophilia, anemia, severe combinedimmunodeficiency). Such protein deficient states are amenable totreatment by replacement therapy, i.e., expression of a protein torestore the normal bloodstream levels of the protein to at least normallevels.

Alternatively, the DNA of interest may encode a polypeptide that iseither normally present in a healthy mammalian subject or which isforeign to the mammalian subject, and which polypeptide is effective intreatment of a condition by expression or over-expression of thepolypeptide. For example, the DNA of interest can encode antimicrobial,antiparasitic, antifungal, or antiviral polypeptides for treatment of amammalian subject having a viral (e.g., human immunodeficiency virus(HIV), Epstein-Barr virus (EBV), herpes simplex virus (HSV), bacterial,fungal, and/or parasitic infection, particularly where the infection ischronic, i.e., persisting over a relatively long period of time. Themethods of the invention may also be used to enhance expression of aprotein present in a normal mammal, or to express a protein not normallypresent in a normal mammal, in order to achieve a desired effect (e.g.,to enhance a normal metabolic process). For example, a secretory glandof a dairy cow may be transformed with DNA encoding bovine growthhormone (BGH) in order to enhance levels of BGH in the bloodstream andenhance milk production.

The DNA of interest is preferably obtained from a source of the samespecies as the mammalian subject to be treated (e.g. human to human),but this is not an absolute requirement. DNA obtained from a speciesdifferent from the mammalian subject can also be used, particularlywhere the amino acid sequences of the proteins are highly conserved andthe xenogeneic protein is not highly immunogenic so as to elicit asignificant, undesirable antibody response against the protein in themammalian host.

Exemplary, preferred DNAs of interest include DNA encoding insulin,growth factors (e.g., growth hormone, insulin-like growth factor-1(IGF-1), platelet-derived growth factor (PDGF), epidermal growth factor(EGF), acidic fibroblast growth factor, basic fibroblast growth factor,or transforming growth factor β), cytokines (e.g., interferon (INF)(e.g., INF-α2b, INF-α2a, INF-αN1, INF-β1b, INF-γ), interleukin (e.g,IL-2, IL-8), or tumor necrosis factor (TNF) (e.g, TNF--α, TNF-β)),clotting factors (e.g., clotting factor VIII), hormones (e.g, GP-1),antimicrobial polypeptides (e.g., antibacterial, antifungal, antiviral,and/or antiparasitic polypeptides), enzymes (e.g., adenosine deaminase),filgastim (Neupogen), hemoglobin, erythropoietin, insulinotropin,imiglucerase, sarbramostim, antigens, tissue plasminogen activator(tPA), urokinase, streptokinase, endothelian, soluble CD4, andantibodies and/or antigen-binding fragments (e.g, FAbs) thereof (e.g.,orthoclone OKT-e (anti-CD3), GPIIb/IIa monoclonal antibody). Preferably,the mammalian subject is a human subject and the DNA expressed encodes ahuman protein.

Table 1 provides a list of exemplary proteins and protein classes whichcan be delivered to the bloodstream of a mammalian subject via themethod of secretory gland cell transformation of the invention.

                  TABLE 1                                                         ______________________________________                                        Exemplary Proteins and Protein Classes for                                      Expression in and Secretion by Secretory Gland Cells                        ______________________________________                                        SPECIFIC EXEMPLARY PROTEINS                                                     insulin            interferon-α2B                                       human growth hormone (hGH) transforming growth factor (TGF)                   erythropoietin (EPO) ciliary neurite transforming factor                       (CNTF)                                                                       clotting factor VIII insulin-like growth factor-1 (IGF-1)                     bovine growth hormone (BGH) granulocyte macrophage colony                      stimulating factor (GM-CSF)                                                  platelet derived growth factor interferon-α2A                           (PDGF)                                                                        clotting factor VIII brain-derived neurite factor (BDNF)                      thrombopoietin (TPO) insulintropin                                            IL-1 tissue plasminogen activator (tPA)                                       IL-2 urokinase                                                                IL-1 RA streptokinase                                                         superoxide dismutase (SOD) adenosine deamidase                                catalase calcitonin                                                           fibroblast growth factor (FGF) arginase                                       (acidic or basic)                                                             neurite growth factor (NGF) phenylalanine ammonia lyase                       granulocyte colony stimulating γ-interferon                             factor (G-CSF)                                                                L-asparaginase pepsin                                                         uricase trypsin                                                               chymotrypsin elastase                                                         carboxypeptidase lactase                                                      sucrase intrinsic factor                                                      calcitonin parathyroid hormone(PTH)-like                                       hormone                                                                      Ob gene product cholecystokinin (CCK)                                         glucagon insulinotrophic hormone                                            EXEMPLARY CLASSES OF PROTEINS                                                   enzymes (e.g., proteases,                                                                        pituitary hormones                                         phospholipases, etc.)                                                         protease inhibitors growth factors                                            cytokines somatomedians                                                       chemokines immunoglobulins                                                    gonadotrophins interleukins                                                   chemotactins interferons                                                      lipid-binding proteins                                                      ______________________________________                                    

Numerous proteins that are desirable for intravenous protein therapy arewell known in the art and the DNA encoding these proteins has beenisolated. For example, the sequence of the DNAs encoding insulin, humangrowth hormone, intrinsic factor, clotting actor VIII, anderythropoietin are available from Genbank and/or have been described inthe scientific literature (e.g., human clotting factor VIII gene:Gitschier et al., Nature 312:326-330, 1984; Wood et al., Nature312:330-337, 1984; human intrinsic factor: Hewitt et al., Genomics10:432-440, 1991). Moreover, proteins commonly used in treatments can beused in the procedures of the present invention. Such proteins aredisclosed in, for example, the Physicians' Desk Reference (1994Physicians' Desk Reference, 48th Ed., Medical Economics Data ProductionCo., Montvale, N.J.; incorporated by reference) and can be dosed usingmethods described in Harrison's Principles of Internal Medicine and/orthe AMA "Drug Evaluations Annual" 1993, all incorporated by reference.

Where the DNA encoding a protein of interest has not been isolated, thiscan be accomplished by various, standard protocols well known to thoseof skill in the art (see, for example, Sambrook et al., ibid; Suggs etal., Proc. Natl. Acad. Sci. USA 78:6613-6617, 1981; U.S. Pat. No.4,394,443; each of which are incorporated herein by reference withrespect to identification and isolation of DNA encoding a protein ofinterest). For example, genomic or cDNA clones encoding a specificprotein can be isolated from genomic or cDNA libraries usinghybridization probes designed on the basis of the nucleotide or aminoacid sequences for the desired gene. The probes can be constructed bychemical synthesis or by polymerase chain reaction (PCR) using primersbased upon sequence data to amplify DNA fragments from pools orlibraries (U.S. Pat. Nos. 4,683,195 and 4,683,202). Nucleotidesubstitutions, deletions, additions, and the like can also beincorporated into the polynucleotides, so long as the ability of thepolynucleotide to hybridize is not substantially disrupted. (Sambrook etal. ibid). The clones may be expressed or the DNA of interest can beexcised or synthesized for use in other constructs. If desired, the DNAof interest can be sequenced using methods well known in the art.

It may also be desirable to produce altered forms of the therapeuticproteins that are, for example, protease resistant or have enhancedactivity relative to the wild-type protein. For example, where thetherapeutic protein is a hormone, it may be desirable to alter theprotein's ability to form dimers or multimeric complexes. For example,insulin may be modified so as to prevent its dimerization has a morerapid onset of action relative to wild-type, dimerized insulin.

Vectors for Delivery of the DNA of Interest to the Secretory Gland Cell

The vectors for delivery of the DNA of interest can be either viral ornon-viral, or may be composed of naked DNA admixed with an adjuvant suchas viral particles (e.g, adenovirus) or cationic lipids or liposomes. An"adjuvant" is a substance that does not by itself produce the desiredeffect, but acts to enhance or otherwise improve the action of theactive compound. The precise vector and vector formulation used willdepend upon several factors such as the secretory gland targeted forgene transfer.

Non-viral vectors

The DNA of interest may be administered using a non-viral vector."Non-viral vector" as used herein is meant to include naked DNA,chemical formulations containing naked DNA (e.g, a formulation of DNAand cationic compounds (e.g., dextran sulfate)), and naked DNA mixedwith an adjuvant such as a viral particle (i.e., the DNA of interest isnot contained within the viral particle, but the transformingformulation is composed of both naked DNA and viral particles (e.g.,adenovirus particles) (see, e.g., Curiel et al. 1992 Am. J. Respir. CellMol. Biol. 6:247-52). Thus "non-viral vector" can include vectorscomposed of DNA plus viral particles where the viral particles do notcontain the DNA of interest within the viral genome.

In one preferred embodiment, the formulation comprises viral particleswhich are mixed with the naked DNA construct prior to administration.Preferably, about 10⁸ to 10¹⁰ viral particles (preferably about 1×10¹⁰to 5×10¹⁰, more preferably about 3×10¹⁰ particles) are mixed with thenaked DNA construct (about 5 μg to 50 μg DNA, more preferably about 8 μgto 25 μg DNA) in a total volume of about 100 μl. Preferably the viralparticles are adenovirus particles (Curiel et al., 1992 supra).

Alternatively or in addition, the DNA of interest can be complexed withpolycationic substances such as poly-L-lysine or DEAC-dextran, targetingligands, and/or DNA binding proteins (e.g, histones). DNA- orRNA-liposome complex formulations comprise a mixture of lipids whichbind to genetic material (DNA or RNA) and facilitate delivery of thenucleic acid into the cell. Liposomes which can be used in accordancewith the invention include DOPE (dioleyl phosphatidyl ethanol amine),CUDMEDA (N-(5-cholestrum-3-β-ol 3-urethanyl)-N',N'-dimethylethylenediamine).

For example, the naked DNA can be administered in a solution containingLipofectin™ LTI/BRL) at a concentrations ranging from about 2.5% to 15%volume: volume, preferably about 6% to 12% volume:volume. Preferredmethods and compositions for formulation of DNA for delivery accordingto the method of the invention are described in U.S. Pat. No. 5,527,928,incorporated herein by reference.

The DNA of interest can also be administered as a chemical formulationof DNA or RNA coupled to a carrier molecule (e.g., an antibody or areceptor ligand) which facilitates delivery to host cells for thepurpose of altering the biological properties of the host cells. By theterm "chemical formulations" is meant modifications of nucleic acids toallow coupling of the nucleic acid compounds to a carrier molecule suchas a protein or lipid, or derivative thereof. Exemplary protein carriermolecules include antibodies specific to the cells of a targetedsecretory gland or receptor ligands, i.e., molecules capable ofinteracting with receptors associated with a cell of a targetedsecretory gland.

Viral vectors

In general, viral vectors used in accordance with the invention arecomposed of a viral particle derived from a naturally-occurring viruswhich has been genetically altered to render the virusreplication-defective and to express a recombinant gene of interest inaccordance with the invention. Once the virus delivers its geneticmaterial to a cell, it does not generate additional infectious virus butdoes introduce exogenous recombinant genes into the cell, preferablyinto the genome of the cell.

Numerous viral vectors are well known in the art, including, forexample, retrovirus, adenovirus, adeno-associated virus, herpes simplexvirus (HSV), cytomegalovirus (CMV), vaccinia and poliovirus vectors.Retroviral vectors are less preferred since retroviruses requirereplicating cells and secretory glands are composed of mostly slowlyreplicating and/or terminally differentiated cells. Adenovirus is apreferred viral vector since this virus efficiently infects slowlyreplicating and/or terminally differentiated cells. The viral vector maybe selected according to its preferential infection of the targetedsecretory gland (e.g., where the secretory gland is a salivary gland,the viral vector may be derived from an attenuated (i.e., does not causesignificant pathology or morbidity in the infected host, e.g, the virusis nonpathogenic or causes only minor disease symptoms) and/orreplication-deficient mumps virus or other attenuated and/orreplication-deficient virus which is substantially specific for salivarygland cells).

Where a replication-deficient virus is used as the viral vector, theproduction of infective virus particles containing either DNA or RNAcorresponding to the DNA of interest can be produced by introducing theviral construct into a recombinant cell line which provides the missingcomponents essential for viral replication. Preferably, transformationof the recombinant cell line with the recombinant viral vector will notresult in production of replication-competent viruses, e.g., byhomologous recombination of the viral sequences of the recombinant cellline into the introduced viral vector. Methods for production ofreplication-deficient viral particles containing a nucleic acid ofinterest are well known in the art and are described in, for example,Rosenfeld et al., Science 252:431-434, 1991 and Rosenfeld et al., Cell68:143-155, 1992 (adenovirus); U.S. Pat. No. 5,139,941 (adeno-associatedvirus); U.S. Pat. No. 4,861,719 (retrovirus); and U.S. Pat. No.5,356,806 (vaccinia virus). Methods and materials for manipulation ofthe mumps virus genome, characterization of mumps virus genesresponsible for viral fusion and viral replication, and the structureand sequence of the mumps viral genome are described in Tanabayashi etal., J. Virol. 67:2928-2931, 1993; Takeuchi et al., Archiv. Virol.,128:177-183, 1993; Tanabayashi et al., Virol. 187:801-804, 1992; Kawanoet al., Virol., 179:857-861, 1990; Elango et al., J. Gen. Virol.69:2893-28900, 1988.

Conditions or Diseases Amenable to Treatment Using the Method of theInvention

Various disease conditions are amenable to treatment using the methodsof the invention. One skilled in the art can recognize the appropriateprotein which should be produced by the invention for treating specificdisease conditions. Exemplary diseases which are amenable to treatmentusing the subject invention, and exemplary, appropriate proteins whichcan be used in treating these diseases, are shown in Table 2.

                  TABLE 2                                                         ______________________________________                                        Exemplary Disease Conditions Amenable to                                        Treatment Using the Invention                                               ______________________________________                                        Enzyme Deficiency                                                                             Endotoxic Shock/Sepsis                                          Adenosine deaminase.sup.1 Lipid-binding protein (LBP)                         Purine nucleotide phosphorylase                                               Galactosidase                                                                 β-glucuronidase                                                          Antioxidants for Cancer Therapy Anemia                                        Superoxide dismutase Erythropoietin                                           Catalase                                                                      Cancer Growth Factors (for use in wound                                       α-Interferon healing, induction of red blood cell                       γ-Interferon formation, etc.)                                           α-IL1 Epidermal growth factor                                           Phenylalanine ammonia lyase G-CSF                                             Arginase γ-Interferon                                                   L-asparaginase Transforming growth factor                                     Uricase Erythropoietin                                                        Granulocyte colony stimulating Thrombopoietin                                 factor (G-CSF) Insulin-like growth factor-1                                   Monoclonal antibodies Insulin                                                 Tissue necrosis factor Human growth hormone                                   Cardiovascular Disease Diabetes                                               Tissue plasminogen activator Insulin                                          Urokinase (native or chimeric) Glucagon                                       α.sub.1 -antitrypsin Insulinotrophic hormone                            Antithrombin-III Clotting disorders                                           Other proteases or protease Clotting factor VIII                              inhibitors                                                                    Apolipoproteins (particularly                                                 B-48)                                                                         Circulating Scavenger Receptor                                                APO A1.sup.2                                                                  Obesity and Feeding Gastrointestinal and Pancreatic                           Ob gene product Deficiencies                                                  Cholecystokinin (CCK) Pepsin (for esophageal reflux)                           Trypsin                                                                      Bone diseases Chymotrypsin                                                    Calcitonin Elastase                                                           PTH-like hormone Carboxypeptidase                                              Lactase (for lactose deficiency)                                              Sucrase; Intrinsic Factor                                                     (pernicious anemia)                                                        Organ-Specific Autoimmune diseases (target of antibody in parentheses)         Myasthenia gravis (acetylcholine receptors)                                   Graves' disease (thyroid-stimulating hormone receptor)                        Thyroiditis (thyroid, peroxidase)                                             Insulin-resistant diabetes with acanthosis nigricans or with ataxia           telangiectasia (Insulin receptor)                                             Allergic rhinitis, asthma (Beta.sub.2 -adrenergic receptors)                  Juvenile insulin-dependent diabetes (insulin, GAD65)                          Pernicious anemia (gastric parietal cells, vitamin B.sub.12 binding site     of                                                                             intrinsic factor)                                                             Addison's disease (adrenal cells)                                             Idiopathic hypoparathyroidism (parathyroid cells)                             Spontaneous infertility (sperm)                                               Premature ovarian failure (interstitial cells, corpus luteum cells)           Pemphigus (intercellular substance of skin and mucosa)                        Bullous pemphigoid (basement membrane zone of skin and mucosa)                Primary biliary cirrhosis (mitochondria)                                      Autoimmune hemolytic anemia (erythrocytes)                                    Idiopathic thrombocytopenic purpura (platelet)                                Idiopathic neutropenia (neutrophils)                                          Vitiligo (melanocytes)                                                        Osteosclerosis and Meniere's disease (type II collagen)                       Chronic active hepatitis (nuclei of hepatocytes)                              Systemic Autoimmune Diseases (defect/organ affected in parentheses)           Goodpasture's syndrome (basement membranes)                                   Rheumatoid arthritis (γ-globulin, EBV-related antigens, collagen        types II and III)                                                             Sjogren's syndrome (γ-globulin, SS-A (Ro), SS-B (La))                   Systemic lupus erythematosus (nuclei, double-stranded DNA,                    single-stranded DNA, Sm                                                       ribonucleoprotein, lymphocytes, erythrocytes, neurons, γ-globulin)      Scleroderm (nuclei, Scl-70, SS-A(Ro), SS-B (La), centromere)                  Polymyositis (nuclei, Jo-1, PL-7, histadyl-tRNA or threonyl-tRNA              synthetases, PM-1, Mi-2)                                                      Rheumatic fever (myocardium heart valves, choroid plexus)                    ______________________________________                                         .sup.1 For treatment of severe combined immunodeficiency                      .sup.2 Converts lowdensity lipoproteins to highdensity lipoproteins      

Transformation of Secretory Gland Cells

The DNA of interest-containing vector (i.e., either a viral or non-viralvector (including naked DNA)) is introduced into the secretory gland invivo via the duct system (i.e., by retrograde ductal injection, whichmay be accomplished by perfusion (i.e., continuous injection), or by asingle, discontinuous injection). Retrograde ductal injection may beaccomplished in the pancreas and liver by endoscopic retrogradechalangio-pancreatography (ECRP). Ductal administration provides severaladvantages. Because the vector is presented to the cells from "outside"the body (from the lumen), the immunological and inflammatory reactionsthat are commonly observed as a result of the administration oftransforming formulations and their adjuvants into blood andinterstitial fluid may be avoided.

Moreover, the cells of secretory glands form a monolayer that enclosesthe duct system. As a consequence, virtually all of the cells of theglands can be accessed by a single administration into the duct. In thisway it is possible to transfect large masses of cells in a relativelysimple manner with a single procedure. The DNA of interest can thus alsobe administered without substantial dilution (it is only diluted by thefluid in the duct system) and without the need to develop organ specifictargeting signals. In contrast, intravenous administration necessarilygreatly dilutes the material and requires that it be targeted to theorgan of interest in some fashion.

The amount of DNA to transform a sufficient number of secretory glandcells and provide for expression of therapeutic levels of the proteincan be readily determined using an animal model (e.g., a rodent (mouseor rat) or other mammalian animal model) to assess factors such as theefficiency of transformation, the levels of protein expression achieved,the susceptibility of the targeted secretory gland cells totransformation, and the amounts of DNA required to transform secretorygland cells.

The precise amount of DNA administered will vary greatly according to anumber of factors including the susceptibility of the target cells totransformation, the size and weight of the subject, the levels ofprotein expression desired, and the condition to be treated. Forexample, the amount of DNA introduced into a secretory gland of a humanis generally from about 1 μg to 200 mg, preferably from about 100 μg to100 mg, more preferably from about 500 μg to 50 mg, most preferablyabout 10 mg. Specifically, the amount of DNA introduced into thepancreas of a human is, for example, generally from about 1 μg to 100mg, preferably about 100 μg to 10 mg, more preferably from about 250 μgto 5 mg, still more preferably from about 500 μg to 1.5 mg, mostpreferably about 1 mg. The amount of DNA introduced into the salivarygland of a human is, for example, generally from about 2.5 μg to 30 mg,more preferably from about 25 μg to 3 mg, still more preferably fromabout 100 μg to 1 mg, most preferably about 250 μg. The amount of DNAintroduced into the liver of a human is, for examples, generally fromabout 10 μg to 500 mg, more preferably from about 100 μg to 300 mg,still more preferably from about 150 μg to 100 mg, most preferably about1 mg.

Generally, the amounts of DNA for human therapy according to theinvention can be extrapolated from the amounts of DNA effective fortherapy in an animal model. For example, the amount of DNA for therapyin a human is roughly 100 times the amount of DNA effective in therapyin a rat. The amount of DNA necessary to accomplish secretory gland celltransformation will decrease with an increase in the efficiency of thetransformation method used.

Concurrent Transformation of Multiple Secretory Glands

In a preferred embodiment of the invention, at least two secretoryglands are transformed according to the methods of the invention. Anytwo secretory glands can be transformed concurrently. For example, theDNA of interest can be administered to both the pancreas and the liver,or to both the salivary gland and the pancreas, or to both the salivarygland and the liver, or to all three. Preferably, cells of the pancreasand the liver are concurrently transformed.

Concurrent transformation of the secretory glands can be carried outseveral hours to several days apart or, preferably, simultaneously(i.e., DNA is introduced into the two secretory glands during the sameprocedure. For example, where the liver and pancreas are to beconcurrently transformed, the DNA formulation can be introducedsimultaneously via a common duct, or separately (e.g., first via thepancreatic duct with occlusion of the hepatic duct, then vice versa).

Concurrent transformation of at least two or more secretory glands canadvantageously provide higher levels of expression of the polypeptide ofinterest in a secretory gland tissue and/or in the bloodstream and can,unexpectedly, provide for synergy between the organs (e.g., to providefor higher levels of tissue expression in a secretory gland than whenthe secretory gland is transformed alone). For example, concurrenttransformation of the pancreas and the liver results in increased levelsof tissue expression in the pancreas relative to tissue levels inpancreas when it is transformed alone.

Moreover, the liver releases the polypeptide of interest in a continuousfashion that is not regulated by hormonal stimulation. The pancreasprovides a relatively lower level of constitutive secretion and storesmost of the polypeptide of interest and only releases large amountsafter stimulation (e.g., after the individual eats). Therefore,transformation of both liver and pancreas has the advantage of providingboth constitutive secretion primarily form the liver, andhormonally-regulated secretion from the pancreas.

Intravenous protein therapy by transformation of salivary gland,pancreatic, and liver cells

Secretory glands transformed according to the invention facilitate highlevels expression of a DNA of interest, particularly where the DNA ofinterest is operably linked to a strong eukaryotic promoter (e.g., CMV,MMTV). The expressed protein is then secreted at high levels into thebloodstream. The protein so expressed and secreted is thus useful intreating a mammalian subject having a variety of conditions.

In a preferred embodiment, the proteins are secreted into thebloodstream at levels sufficient for intravenous protein therapy. Forexample, the amount of a specific protein normally released into theblood from the pancreas can be substantial, e.g., a specific proteinthat is released into the bloodstream can be as much as 25% of theamount of duct-directed secretion of that specific protein. This amountsto as much as 1-2 mg of protein/gram of tissue being directed into theblood per hour.

Bloodstream levels of the therapeutic protein may be enhanced by severaldifferent methods. For example, bloodstream levels can be enhanced byincreasing the overall level of expression of the desired protein, e.g.,by integration of multiple copies of the DNA of interest into the genomeof the target cells, by operably linking a strong promoter (e.g., apromoter from CMV) and/or enhancer elements to the DNA of interest inthe construct, or by transformation of a greater number of target cellsin the subject (e.g., by administration of multiple doses of thetransforming material).

Secretion of the therapeutic protein into the bloodstream can also beenhanced by incorporating leader sequences, amino acid sequence motifs,or other elements that mediate intravenous-directed secretion into thesequence of the therapeutic protein. For example, the DNA of interestcan be engineered to contain a secretion signal that directs secretionof the protein primarily into the bloodstream, thereby increasing theamount of the protein produced in the secretory gland that reaches inthe bloodstream. Intravenous-directed secretion signals can beidentified by, for example, site-directed mutagenesis of DNA encoding abloodstream-targeted protein (e.g., insulin). The mutants can bescreened by expression of the mutated DNA in secretory gland cells andsubsequently determining the ratio of, for example, salivary tointravenous expression.

Alternatively, intravenous-directed secretion signals can be identifiedby constructing recombinant, chimeric proteins composed of, for example,a putative intravenous secretion signal inserted into a saliva-directedprotein. Intravenous secretion signals would then be identified by theirability to re-direct expression of the saliva-directed protein into thebloodstream. Putative intravenous secretion signals and duct systemsecretion signals can also be identified by comparison of DNA and aminoacid sequences of proteins which are preferentially secreted into thebloodstream. Areas of homology or common motifs among the proteins couldthen be tested as described above.

Overall secretion from secretory glands can be augmented by hormonalstimulation. For example, where the protein is primarily secreted intothe duct system and is secreted at lower levels into the bloodstream,hormonal stimulation enhances intravenous secretion as well as secretioninto the duct. Thus, therapeutically effective levels of the protein thebloodstream may be achieved or enhanced by administration of anappropriate, secretory gland specific hormone. For example, secretorygland secretion can be enhanced by administration of a cholinergicagonist such as acetyl-β-methyl choline, or can be augmented or furtheraugmented by control of diet (i.e., eating stimulates pancreatic andsalivary gland secretion). Thus, because eating a meal can elicit asecretory response, adjustment of meals (e.g., frequency of meals and/oramounts eaten) can be used as a dosing mechanism for delivery of thedesired protein, and can be accomplished without administration ofadditional protein-encoding DNA.

Bloodstream-directed secretion can also be regulated at either the levelof transcription, translation, or secretion. Transcriptional regulationinvolves the timing and level of transcription directed from the DNA ofinterest, while translational regulation involves the production ofpolypeptides from transcribed RNA. Secretory regulation involves therelease of polypeptides from the cell (e.g., from secretory cells inwhich the polypeptides to be secreted are stored within intracellularvacuoles). Methods for providing transcriptional and/or translationalregulation of a DNA of interest are well known in the art (e.g,transcriptional regulation through the use of inducible promoters).

Secretory regulation can be achieved by, for example, administration ofa hormone that elicits a secretory response in the desired secretorygland, or by activity that stimulates production of such hormone(s)(e.g., eating to stimulate pancreatic secretion). Unlike regulation atthe level of transcription or translation, which can take many hours tobecome effective, regulation of secretion occurs within minutes afterstimulation. Moreover, endocrine secretion from the pancreas andsalivary glands is stimulated by hormones and neurotransmitters that arenatural components of the feeding response; thus feeding itself can actas a dosing mechanism.

The actual number of transformed secretory gland cells required toachieve therapeutic levels of the protein of interest will varyaccording to several factors including the protein to be expressed, thelevel of expression of the protein by the transformed cells, the rate ofprotein secretion, the partitioning of the therapeutic protein betweenthe gastrointestinal tract and the bloodstream, and the condition to betreated. For example, the desired intravenous level of therapeuticprotein can be readily calculated by determining the level of theprotein present in a normal subject (for treatment of a proteindeficiency), or by determining the level of protein required to effectthe desired therapeutic result.

Application of the Method of the Invention to Achieve Euglycemia in aDiabetic Syndrome

In another preferred embodiment of the invention, pancreatic cells aretransformed using insulin-encoding DNA to provide for expression andsecretion of insulin into the bloodstream of a mammalian subject.Transformation of pancreatic cells with insulin encoding DNA not onlyprovides for regulated expression of insulin in a mammalian subject, butalso provides for maintenance of a euglycemic state (i.e., normal bloodglucose levels) in diabetic subjects for extended periods of time (e.g.,up to 6 to 7 days post transformation). Thus, not only is the exocrinepancreas secreting insulin to reduce blood sugar, but regulating itssecretion so that blood levels are maintained at normal levels, e.g, areregulated. Thus, pancreatic transformation with insulin-encoding DNA canbe used in the therapy of individuals having a disease or conditionassociated with elevated blood glucose levels (e.g., diabetes (e.g.,type I or type II diabetes), and hyperglycemia). This aspect of theinvention may be applied to regulate levels of other proteins in thebloodstream.

Assessment of Protein Therapy

The effects of expression of the protein encoded by the DNA of interestfollowing in vivo transfer of the DNA of interest can be monitored in avariety of ways. Generally, a sample of blood from the subject can beassayed for the presence of the therapeutic protein. Appropriate assaysfor detecting a protein of interest in blood samples are well known inthe art. For example, a sample of blood can be tested for the presenceof the polypeptide using an antibody which specifically binds thepolypeptide in an ELISA assay. This assay can be performed eitherqualitatively or quantitatively. The ELISA assay, as well as otherimmunological assays for detecting a polypeptide in a sample, aredescribed in Antibodies: A Laboratory Manual (1988, Harlow and Lane,eds. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

Alternatively, or in addition, the efficacy of the polypeptide therapycan be assessed by testing a sample of blood for an activity associatedwith the polypeptide (e.g., an enzymatic activity). Furthermore, theefficacy of the therapy using the methods of the invention can beassessed by monitoring the condition of the mammalian subject forimprovement. For example, where the polypeptide is erythropoietin, thesubject's blood is examined for iron content or other parametersassociated with anemia.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tocarry out the invention and is not intended to limit the scope of whatthe inventors regard as their invention. Efforts have been made toensure accuracy with respect to numbers used (e.g., amounts,temperatures, etc.), but some experimental error and deviation should beaccounted for. Unless indicated otherwise, parts are parts by weight,molecular weight is weight average molecular weight, temperature is indegrees Centigrade, and pressure is at or near atmospheric.

EXAMPLE 1 In vivo gene transfer of DNA encoding human growth hormone byretrograde injection of DNA into the pancreas

Four constructs for expression of human growth hormone (hGH) wereprepared using techniques well known in the art (see, for example,Sambrook et al. ibid). The first construct, pFGH, contains the genomichGH DNA sequence inserted in the commercially available vectorpBLUESCRIPT SK+™ (Stratagene, LaJolla Calif.) (FIG. 1). Because the hGHcoding sequence is not linked to a promoter, this vector provides for noor only low-level hGH expression. Thus, the pFGH construct serves as anegative control for hGH expression in the pancreas. The secondconstruct, pFGH.CMV, was constructed by operably inserting the promoterfrom the immediate early gene of human CMV upstream of the genomic hGHsequence of the pFGH vector (FIG. 2). The third construct, pFGH.chymo,was constructed by operably inserting the rat chymotrypsin B genepromoter upstream of the genomic hGH sequence of the pFGH vector (FIG.3). The fourth construct, pFGH.RSV, was constructed by operablyinserting the promoter from the long terminal repeat (LTR) of RSVupstream of the genomic hGH sequence of the pFGH vector.

Each of the four vectors was used to transfect the pancreas ofapproximately 300 g adult male, Sprague-Dawley rats (pFGH+lipofectin, 4rats; pFGH.chymo+lipofectin, 4 rats; pFGH.RSV+lipofectin, 4 rats;pFGH.CMV+lipofectin, 10 rats; pFGH.CMV without lipofectin, 7 rats;negative control (no DNA, no lipofectin), 3 rats). Pancreatictransfection was accomplished by first anesthetizing the rats andperforming a laparotomy to expose the duodenum. The pancreas and theassociated common bile duct were identified, and the common bile ductwas cannulated either extraduodenally or through the papilla of Vater.The hepatic duct was occluded, and 100 μl of phosphate-buffered saline(PBS) containing one of the four vectors, or 100 μl of PBS alone as anegative control, were slowly injected or infused into the pancreaticduct in a retrograde direction. The vector-containing solutions werecomposed of 8 μg DNA per 100 μl in PBS, either with or without 6%lipofectin, a cationic lipid used to increase transfection efficiency.The solution was left in place for 5 min before secretory flow wasallowed to resume and hepatic duct blockage removed. The catheter wasleft in place and inserted into the duodenum through a small hole toensure adequate biliary and pancreatic flow post-operatively. Theabdomen was then closed with sutures. The animals recovered fully andrapidly from the surgery without obvious side effects. This transfectionmethod provides direct access of the vector to over 90% of thepancreatic gland cells.

At 48 hr after surgery, a blood sample was obtained to measure serum hGHlevels, and the rats were sacrificed. At autopsy, the pancreas of bothcontrol and test rats appeared normal, and exhibited no gross ormicroscopic pathology.

The pancreas was dissected free from the mesenteric surface and washomogenized in cold 0.2 M (pH 8.0) sodium phosphate buffer (1:10 w/v)containing protease inhibitors aprotinin, leupeptin, pepstatin, andPEFABLOC SC™. Homogenization was completed by shearing after 10 passeswith a motorized pestle at approximately 4000 rpm in a glasshomogenizer. The homogenate was then centrifuged at 1000 g for 15 min.The supernatant was collected and stored at -80° C. until analysis. Thelevels of hGH in the serum and pancreatic protein samples were measuredusing the hGH radioimmune assay (Nichols Institute). Each assay wasperformed in duplicate and compared to a set of control samples.

Rats injected with the pFGH.CMV vector expressed higher levels of hGH inthe pancreatic tissue (FIG. 4), compared to background levels ofpancreatic hGH expression in rats injected with either no DNA (PBSalone) or the pFGH vector (hGH DNA with no promoter). The addition oflipofectin modestly increased hGH expression in rats injected with thepFGH.CMV construct. In addition, rats transfected with the pFGH.CMVvector secreted hGH in the serum at levels increased relative tobackground levels and to hGH secretion levels in rats injected witheither control samples (no DNA or pFGH) or with samples containing hGHDNA linked to either the chymotrypsin B or RSV promoters (FIG. 5). InFIG. 6, all data from the above experiments (including all controls andvectors) are analyzed by plotting the hGH serum levels against the hGHtissue levels. This graph shows that higher tissue levels result inhigher levels of secretion into the blood. Thus, retrograde pancreaticinjection of the pFGH.CMV vector successfully transfected pancreaticcells to provide both hGH pancreatic tissue expression and hGH secretioninto the bloodstream.

EXAMPLE 2 In Vivo Transformation of Pancreatic Cells by RetrogradeDuctal Injection of hGH-Encoding DNA and Regulation of hGH Secretion

Eight rats were anesthetized and control blood samples (no DNA) werecollected from the femoral vein of each animal. Pancreatic transfectionwas accomplished by exposing the duodenum by laparotomy and identifyingthe pancreas and the associated common bile duct. The common bile ductwas cannulated either extraduodenally or through the papilla of Vater,and the hepatic duct was occluded. A 1:50 dilution ofreplication-defective human adenovirus (Ad5-di 342) supernatant in 100μl of phosphate-buffered saline (PBS) containing 8 μg of thehGH-encoding plasmid pFGH.CMV (FIG. 2) was slowly infused into thepancreatic duct in a retrograde direction. The solution was left inplace for approximately 5 min before secretory flow was allowed toresume and the hepatic duct blockage removed. The catheter was left inplace and inserted into the duodenum through a small hole to ensureadequate biliary and pancreatic flow post-operatively. The abdomen wasthen closed with sutures. The animals recovered fully and rapidly fromthe surgery without obvious side effects.

At 48 hr after surgery, a blood sample was obtained to measure serum hGHlevels (unstimulated serum levels). The cholinergic agonist McH wasinjected subcutaneously into each rat at 0.8 mg/kg body weight. Bloodsamples were collected from the inferior vena cava of each animal at 15min intervals following McH injection. Serum was separated from theblood of all samples after clotting, and kept at -20° C. prior to assay.

As shown in FIG. 9 (one representative animal) plasma levels of hGHincreased markedly following McH injection, demonstrating that secretionof hGH expressed by transformed pancreatic cells is regulated by agoniststimulation. Moreover, bloodstream-directed secretion of hGH from thetransformed pancreatic cells occurred at relevant, physiological levelsuseful in therapeutic administration (i.e., at the ng/ml level).

EXAMPLE 3 In Vivo Transformation of Salivary Glands by Retrograde DuctalInjection of DNA Encoding Human Growth Hormone

Twelve adult rats weighing approximately 300 g each were anesthetizedwith an intraperitoneal injection of sodium pentobarbital. A totalvolume of 50 μl containing 4 μg of the pFGH.CMV plasmid, which containscDNA encoding human growth hormone (hGH) (FIG. 2), was introduced intoeach submandibular gland of 8 rats by retrograde ductal injection viathe ducts leading from the oral mucosa to the salivary gland. Briefly,both the left and right Wharton's duct were cannulated intraorally withpolyethylene (PE) 10 tubing, and the DNA injected into the duct systemof each gland in a retrograde fashion (4 μg/50 μl of PBS). The materialwas kept in place for two minutes before normal flow was reestablished.

For three of these animals the DNA was mixed in a 6% solution of thecationic lipid Lipofectin (labeled "liposomes") from Life Technologies(Gaithersburg, Md.). For four of these animals, the DNA was mixed with a1:50 dilution of replication-defective human adenovirus (Ad5-di 342)supernatant. Control rats (4 rats) received 50 μl 0.9% saline (control)without plasmid. No significant leakage of material or bleedingoccurred. After 3 hours, the animals were awake, drinking water, andappearing normal.

Approximately 48 hours after cDNA injection, the animals weresacrificed. The right and left submandibular glands were removed andwere homogenized in cold 0.2 M (pH 8.0) sodium phosphate buffer (1:10w/v) containing the protease inhibitors aprotinin, leupeptin, pepstatin,and PEFABLOC SC™. Homogenization was completed by shearing after 10passes with a motorized pestle at approximately 4000 rpm in a glasshomogenizer. The homogenates were centrifuged at 1000 g for 15 min, andthe supernatant collected and stored at -80° C. until analysis. Thelevels of hGH in the protein samples were measured using the hGHradioimmune assay (Nichols Institute). Each assay was performed induplicate and compared to a set of control samples.

Each of the submandibular glands of the rats injected with the pFGH.CMVvector expressed hGH in the salivary gland tissue; hGH expression wasundetectable in the control rats' salivary glands (FIG. 7).

EXAMPLE 4 In Vivo Transformation of Salivary Glands by Retrograde DuctalInjection of hGH-Encoding DNA and Regulation of hGH Secretion

Three adult rats weighing approximately 300 g each were anesthetizedwith an intraperitoneal injection of sodium pentobarbital. A controlblood sample (prior to DNA) was drawn from the femoral vein of eachanimal. A total of 4 μg of the hGH-encoding plasmid pFGH.CMV (FIG. 2) in50 μl, was introduced into each submandibular gland of each rat byretrograde ductal injection via the ducts leading from the oral mucosato the salivary gland as described above in Example 2. No significantleakage of material or bleeding occurred. After 3 hours, the animalswere awake, drinking water, and appearing normal.

Forty-eight hours after cDNA injection, the animals were againanesthetized and a control blood sample was drawn from the femoral veinof each animal (unstimulated serum level). The cholinergic agonistacetyl-β-methyl choline McH) was injected subcutaneously at 0.8 mg/kgbody weight into each animal. Blood samples were collected from thefemoral vein of each animal at 10 min, 20 min, 40 min, and 50 min afterMcH injection. Serum was separated from the blood of all samples afterclotting, and kept at -20° C. prior to assay.

As shown in FIG. 8 (one representative animal), secretion of hGH intothe bloodstream was dramatically increased in response to administrationof McH, peaking at 40 min. Thus, these data demonstrate thatintroduction of hGH-encoding DNA into the salivary gland results inbloodstream-directed secretion of hGH and regulation by cholinergicstimulation. Moreover, regulation is at the level of secretion, nottranscription, since transcriptional regulation would not result inincreased hGH bloodstream levels in such a short period.

EXAMPLE 5 Treatment of Diabetes Mellitus Over a Three Day Period by InVivo Transformation of Pancreatic Cells by Retrograde Ductal Injectionwith Insulin-Encoding DNA

Streptozotocin, which induces diabetes mellitus in rats, wasadministered to 8 male Sprague-Dawley rats (260-280 g) after overnightfasting by intraperitoneal injection in 1 mM citrate buffer (pH 4.5)(Sigma) at 65 mg/kg of body weight. One hour later, animals wereanesthetized with Nembutal and the body cavity opened to expose thegastrointestinal tract. Each animal was given the appropriate DNAconstruct directly by retrograde injection in the pancreatic duct in a100 μl injection volume containing 8 μg DNA plus adenovirus (Ad5-di342)(3×10¹⁰ viral particles) as described above. Test animals (4 rats)received the human insulin-encoding construct pBat16.hInsG1.M2. ThepBAT16.hInsG1.M2 construct (FIG. 10) encodes an insulin gene containinga site-directed mutation of the second protease site to create a furinrecognition site; this construct provides for enhanced expression ofprocessed insulin in non-neuroendocrine cells. In addition, the humanβ-globin first intron replaces the first insulin gene intron which isinefficiently spliced. Control animals (4 rats) received the controlconstruct CMV-GFP, which contains a green fluorescent protein(GFP)-encoding sequence operably linked to a CMV promoter. The animalsrecovered fully and rapidly from the surgery without obvious sideeffects. Body weight and blood glucose were monitored daily for threedays post-injection. Blood glucose was measured by the glucose oxidasemethod (Lifescan, Milpitas, Calif.).

As shown in FIG. 11, treatment of the streptozotocin-induced diabeticrats with the insulin-encoding construct resulted in maintenance ofalmost complete euglycemia for 3 days. In contrast, control animals thatreceived the GFP-encoding construct remained hyperglycemic throughoutthe test period. The data show that introduction of insulin-encoding DNAinto the pancreas results in pancreatic cell transformation, as well assecretion of insulin by the transformed pancreatic cells at levelssufficient to overcome diabetes in an animal model. Moreover, theseresults show that the method of the invention provides regulated andrelatively normal blood glucose levels. Surprising, the exocrinepancreas regulates the release of insulin such that blood sugar levelsare maintained at regulated levels (normally the endocrine pancreas isresponsible for regulation of bloodstream-directed secretion).

EXAMPLE 6 Treatment of Diabetes Mellitus Over a Six Day Period by InVivo Transformation of Pancreatic Cells by Retrograde Ductal Injectionwith Insulin-Encoding DNA

Streptozotocin was administered to 14 rats at 70 mg/kg body weight byintraperitoneal injection to induce diabetes mellitus. The animals werethen anesthetized by intraperitoneal injection of sodium pentobarbital.Two rats did not receive streptozotocin and served as one negativecontrol. Insulin-encoding DNA in the pBAT16.hInsG 1.M2 construct (FIG.10) was administered to 8 of the streptozotocin-injected rats byretrograde ductal injection as described above. Sixstreptozotocin-treated rats received either 100 μl of saline without DNA(2 animals) or a control DNA without the human insulin gene (4 animals)by pancreatic retrograde ductal injection as additional negativecontrols. The animals recovered fully and rapidly from the surgerywithout obvious side effects. Blood samples were collected from thefemoral vein of each animal at 24 hr intervals for 6 days. Human insulinwas measured using a double antibody radioimmunoassay (LincoLaboratories, Saint Louis, Mo.).

As shown in (FIG. 12), blood glucose levels were significantly decreasedin the diabetic rats that received the insulin-encoding DNA (+Strep,+DNA) relative to diabetic the rats that received no DNA (+Strep, NoDNA). Furthermore, these decreased blood glucose levels were observedthroughout the entire 6 day course of the experiment. Thus, these datashow that introduction of insulin-encoding DNA into the pancreas resultsin persistent expression of insulin, and that the insulin expressed bythe transformed pancreatic cells is secreted into the bloodstream andcan function in regulation of blood glucose at levels sufficient toovercome diabetes in an animal model. As shown in FIG. 12, elevatedinsulin levels for such an extended period additionally demonstrateprolonged expression from the DNA introduced into the pancreatic cells.

EXAMPLE 7 In vivo Transformation of Pancreatic Cells by RetrogradeDuctal Injection of Green Fluorescent Protein-Encoding DNA andExpression in Pancreatic Cells

To identify the pancreatic cells that expressed the recombinant protein,DNA encoding green fluorescent protein (GFP) was used to transformpancreatic cells according to the methods of the invention. EGFP cDNAfrom plasmid pEGFP.C2 (Clontech) was inserted into pFOX. The EGFPsequence was modified to contain an SV40 nuclear localization signal,in-frame at the 3' end. This addition allowed for partial nuclearlocalization and facilitated immunohistochemical detection. The CMVimmediate early promoter was positioned upstream of the first intron ofhuman β-globin to create the expression vector pFOX.EGFP.N2.CMV.

After fasting overnight, Male Sprague-Dawley rats (260-280 g) wereanesthetized and the body cavity opened to expose the gastrointestinaltract. The green fluorescent protein (GFP)-encoding constructpFOX.EGFP.N2.CMV was administered to each animal by retrograde injectionin the pancreatic duct in a 100 μl injection volume containing 8 μg DNApremixed with adenovirus (3×10¹⁰ viral particles) as described above.The animals recovered fully and rapidly from the surgery without obviousside effects.

Seventy-two hours post-treatment, the animals were sacrificed, andpancreases were removed and weighed (wet weight). Samples of eachpancreas were fixed in 5% buffered formalin for 24-48 hours at roomtemperature. Fixed tissues were dehydrated and imbedded in paraffin, and5 μm sections were processed for immunohistochemistry using standardtechniques. Endogenous peroxidase was quenched in 0.7% H₂ O₂ /MeOH, andantigen retrieval was performed using Citra solution (Biogenex, SanRamon, Calif.) according to the manufacturers' instructions. Sectionswere preincubated for 30 minutes in 5% goat serum/phosphate-bufferedsaline (PBS), and then incubated overnight at 4° C. with primaryantisera diluted in 5% goat serum/PBS.

The primary antisera were selected from either anti-GFP antisera(1:1500; Clontech, Palo Alto, Calif.), anti-insulin antisera (1:500;Dako, Carpenteria, Calif.), or non-specific rabbit sera (1:1500). Thefollowing day all sections were incubated with biotinylated goatanti-rabbit antiserum (5 μg/ml; Vector, Burlingame, Calif.) for 30minutes at room temperature, and then incubated withstreptavidin-aminohexanol-biotin horseradish peroxidase (HRP) complex(Vectastain-Elite, Vector). Protein was visualized by reaction with theperoxidase substrate 3,3-diamino-benzidine tetrahydrochloride (DAB;Sigma). The color reaction was followed by a brief counter stain in 1%methyl green (Sigma) prior to mounting. Negative controls includedstaining of sections from pancreas not injected with CMV-GFP, andomission of primary antiserum.

Staining for GFP was observed in the pancreas of animals treated withGFP DNA, but not in control animals. GFP expression was restricted toexocrine cells; there was no staining in either ductal or islet cells.Moreover, expression was observed in 0.1-1.0% of exocrine cells.Endogenous insulin was detected in adjacent sections; but GFP expressiondid not co-localize with insulin expression, suggesting that thepancreatic cells primarily transformed are exocrine, not endocrinecells. Under the conditions studied there was no histological indicationof inflammatory infiltration as a consequence of ductal injection of thevector.

These data show that introduction of the DNA construct results insuccessful transformation of pancreatic cells, despite the introductionof the construct against the flow of pancreatic juices and the highconcentrations of DNase in the pancreatic juice. Moreover, these data,combined with the data above showing that transformation of the pancreasresults in bloodstream-directed secretion of the encoded protein, andsuggest that transformation of exocrine pancreatic cells results inbloodstream-directed secretion of the protein encoded by the introducedconstruct. Furthermore, because insulin staining and GFP staining didnot co-localize, introduction of the GFP-encoding construct resulted intransformation of exocrine tissue, which is normally associated withprotein secretion into the gastrointestinal tract, rather than endocrinetissue, which is normally associated with bloodstream-directedsecretion. Despite this, bloodstream-directed secretion was stillobtained at physiologically relevant levels sufficient to treat diabetesmellitus in an animal model as evidenced in the examples above.

EXAMPLE 8 In Vivo Transformation of Liver Cells by Retrograde DuctalInjection of hGH-Encoding DNA and Bloodstream-Directed hGH Secretion

Four rats were anesthetized and control blood samples (no DNA) werecollected from the femoral vein of each animal. Transfection of livercells was accomplished by exposing the duodenum by laparotomy andidentifying the liver and the associated common bile duct. The commonbile duct was cannulated either extraduodenally or through the papillaof Vater. The tubing was advanced to the bifurcation of the hepatic ductin order to prevent injected material from entering the distally locatedpancreatic drainage. A 1:50 dilution of replication-defective humanadenovirus supernatant in 100 μl of phosphate-buffered saline (PBS)containing 8 μg of the hGH-encoding plasmid pFGH.CMV (FIG. 2) or 100 μlof PBS alone (no DNA) were slowly infused into the hepatic duct in aretrograde direction. The solution was left in place for approximately 2min to 5 min before secretory flow was allowed to resume and thepancreatic duct blockage removed. The catheter was left in place andinserted into the duodenum through a small hole to ensure adequatebiliary and pancreatic flow post-operatively. The abdomen was thenclosed with sutures. The animals recovered fully and rapidly from thesurgery without obvious side effects.

Plasma hGH levels were measured 2 days after treatment; the results areshown in FIG. 13. Each data point in FIG. 13 represents themean±standard error of the mean (SEM) for three animals. These datademonstrate that liver cells were transformed with the hGH-encoding DNA.Furthermore, hGH was secreted by the transformed liver cells into thebloodstream at physiologically relevant levels.

EXAMPLE 9 In Vivo Transformation of Pancreatic Cells with hGH-EncodingDNA and Expression in Rat Exocrine Pancreas and Plasma

Following overnight fasting and anesthesia with pentobarbital, theabdominal cavity of the rats was opened and the pancreatic ductcannulated external to the duodenum with PE 10 tubing as describedabove. Eight to twenty-five micrograms of each of pFGH (promoter lessconstruct), pFGH.chymo (construct with the chymotrypsin promoter),pFGH.RSV (construct with the RSV promoter), and pFGH.CMV (construct withthe CMV promoter) was injected in a total volume of 100 μl of PBS intothe pancreas via the pancreatic duct as described above. Immediatelyprior to injection construct samples were optionally premixed witheither Lipofectin (6-12% vol:vol) or adenovirus (3×10¹⁰ viralparticles). The material was kept in the duct for 5 min prior toestablishing normal flow. The abdomen was the closed and the animalsallowed to recover.

Forty-eight hours later the pancreas was harvested, plasma obtained, andhuman growth hormone measured. The animals were anesthetized, bloodsamples taken (either from the femoral vein or inferior vena cava), andthe transfected tissue removed. The tissue was homogenized in PBScontaining 5 mM Na2HPO₄ (pH 7.8) at a tissue to fluid ratio of 1:10using a motorized mortar and pestle. Large particulate material in thehomogenate was removed by sedimentation at 10,000× g for 30 minutes, andthe supernatant assayed for the protein of interest. The results areshown in FIGS. 14-17. All data shown are the mean±the SEM.

The effects of the various promoters upon tissue expression andsecretion of hGH into the bloodstream are shown in FIGS. 14 and 15,respectively. In these experiments, the constructs were mixed withlipofection prior to administration. Of the promoters tested, the CMVpromoter was by far the most effective, and produced high levels of hGHin tissue (in the range of 150 ng/g tissue wet weight) when compared toeither promoter less controls, or plasmids containing RSV andchymotrypsin promoters (FIG. 14). The cationic lipid adjuvant Lipofectinincreased expression by about 50%, and pre-mixing the plasmid withadenovirus enhanced tissue expression five fold (FIG. 15). Expression ofhGH at 24, 48 or 72 hours after injection was similar under allconditions studied.

As shown in FIGS. 16 and 17, hGH was secreted into plasma. Plasmidscontaining the CMV promoter increased circulating levels of hGH fivetimes above background (FIG. 16). With plasmid alone, plasma hGHconcentrations in the range of 60 to 80 pg/ml were routinely observed.Premixing the plasmids with adjuvants also increased circulating hGHlevels (FIG. 17). Lipofectin increased plasma levels by an additional50%, and adenovirus by 75%, when compared to plasmid alone.

EXAMPLE 10 Comparison of hGH Secretion by Rat Liver, Pancreas, andCombined Liver and Pancreas Transformed with hGH-Encoding DNA

Eight micrograms of the pFGH.CMV construct premixed with adenovirus asdescribed above in Example 9, was injected into the ducts of either theliver, the pancreas, or both organs of the same animal. Where only theliver or the pancreas was transformed (liver alone or pancreas alone),the DNA was introduced according to the methods described above. Whereboth the liver and pancreas were transformed, the DNA-containingformulation was introduced into the hepatic duct first, and then thetubing partially withdrawn to provide access to the pancreatic ductsystem. A temporary ligature was then placed around the hepatic duct toprevent the second infusion from entering the parenchyma of the liver.Thus, animals in which both the pancreas and liver were transformedreceived two doses of the DNA-containing formulation. Plasma hGH levelswere measured two days later.

In animals having transformed liver (liver alone) or pancreas (pancreasalone), hGH was expressed in liver or pancreatic tissue, respectively,and hGH detected in plasma under both circumstances. Tissue levels inliver when transformed alone were far lower than in the pancreas whentransformed alone (less than 1 ng/g, as compared to about 500 ng/g), buthGH concentration in plasma of animals in which only the liver wastransformed was nonetheless comparable to hGH plasma levels in animalshaving only the pancreas transformed (in the range of 0.15 ng/ml; FIG.18). These results are consistent with the observation that, in contrastto the exocrine cells of the pancreas and salivary glands, hepatocytessecrete most of what they produce soon after synthesis.

When pancreas and liver were both transfected, plasma levels were higherthan seen when the glands were treated individually (nearly 0.3ng/ml)--a value approximately equal to the sum of that observed for thetwo organs separately. Surprisingly, transformation of both liver andpancreas resulted in tissue levels in the pancreas being significantlyincreased relative to tissue levels in the pancreas when the pancreaswas transformed alone (FIG. 19).

EXAMPLE 11 Human growth hormone (hGH) expression in rat salivary gland.

Four micrograms of the pFGH.CMV construct, premixed with eitherLipofectin or adenovirus, was injected into each submandibular gland viaretrograde ductal injection (via Wharton's duct) as described above. Twodays later, each gland was harvested and hGH content was measured.

As shown in FIG. 20, tissue levels of hGH averaged about 50 ng/g tissuewet weight. Plasma hGH levels were in the 20-40 pg/ml range. As in thepancreas, the addition of adenovirus increased tissue hGH levels, inthis case to 100 ng/g (FIG. 20).

EXAMPLE 12 Stimulation of Human growth hormone (hGH) secretion

Even when exocrine secretory cells store large amounts of protein, suchas after a period of fasting, they secrete these proteins at a low rateunder unstimulated conditions (i.e. basal or constitutive secretion).Greater rates are achieved when exogenous stimulants (e.g., hormonalstimulants and/or stimulation associated with eating) are applied. Todetermine whether secretion of the engineered protein would be enhancedduring feeding, pancreatic secretion was stimulated with a secretorystimulant. For these experiments we used animals in which both pancreasand liver were transfected. Eight micrograms of the pFGH.CMV constructwere injected into ducts of both the pancreas and liver of four rats asdescribed above. A blood sample was taken prior to injection as acontrol. Two days after transfection, a second control blood sample wastaken and the rats were treated with the cholinergic agonist,acetyl-β-methylcholine (McH) (0.8 mg/kg body weight).

As shown in FIG. 21, hGH secretion was increased three fold within 30minutes of stimulation, with plasma levels approaching 1.0 ng/ml.Similar enhancement of hGH secretion was observed when either thepancreas was studied alone, or when the salivary glands were studiedalone. These data show that hGH secretion is enhanced by stimulationwith a cholinergic agonist. Thus secretion of hGH is regulated in amanner similar to secretion of endogenous proteins.

Although the concentration of hGH in plasma was correlated to the levelof hormone in the pancreas (r=0.55, p<0.01, n=41), at high tissuelevels, plasma concentration was not linearly proportional to tissuecontent. For example, addition of adenovirus to the hGH vector produceda five fold increase in tissue levels relative to the plasmid alone(FIG. 20), but only about a two fold increase in plasma concentration(see, e.g., FIGS. 16 and 17, Example 9 above). This lack ofproportionality indicates that it is not the concentration of product inthe cells alone that determines the rate of secretion into blood, butthat at high tissue levels, secretion is limited by other factors. Thisresult is similar to what is observed for endogenous protein secretionand suggests that secretion of the engineered protein is regulated inmuch the same manner.

EXAMPLE 13 Human Insulin Expression and Secretion in Diabetic RatPancreas

In an attempt to treat a disease state, diabetes mellitus, we expressedhuman insulin in the exocrine pancreas. Fasted experimental and controlanimals received intra-peritoneal streptozotocin (Sigma; 65 mg/kg bodyweight, in 1 mM citrate buffer, pH4.5) on day zero one hour prior toadministration of the insulin-encoding construct. The experimentalanimals subsequently received 8 μg of the insulin plasmid(pBAT16.hInsG1.M2) premixed with adenovirus and injected into thepancreatic duct, also on day zero. The pBAT16.hInsG1.M2 constructcontains the human insulin cDNA linked to a CMV immediate earlypromoter, which is positioned upstream of the first intron of humanβ-globin. The human insulin cDNA was mutated to convert the secondprotease site, between peptides C and A, to a furin recognition site.This allows for correct proteolytic processing of mature insulin innon-endocrine cells.

Plasma insulin and glucose levels were determined for up to six days.Plasma glucose levels in diabetic rats (n=3), and diabetic rats treatedwith the pBAT16.hInsG1.M2 plasmid (n=3), measured over a three dayperiod, are shown in FIG. 22. Plasma insulin levels in diabetic rats(n=3), and diabetic rats treated with the pBAT16.hInsG1.M2 plasmid(n=3), measured over a three day period, are shown in FIG. 23. Plasmaglucose levels in individual diabetic (n=3) and pBAT16.hInsG1.M2plasmid-treated diabetic rats (n=3), measured over a six day period, areshown in FIG. 24.

As a consequence of streptozotocin administration, blood glucose levelsrose from the normal level of 100 mg/dl to 300-400 mg/dl within 24 hoursand remained elevated for the duration of the study (FIG. 22). Treatmentwith the human insulin plasmid reduced blood glucose levels in diabeticrats to the normal range (FIGS. 22 and 24), and concentrations ofinsulin remained near pre-treatment values (FIG. 23). Blood glucoselevels were euglycemic for the duration of the study (6 days; FIG. 24).Animals transfected with a control plasmid remained diabetic (data notshown). These data show that regulation of insulin secretion in responseto feeding was effective.

EXAMPLE 14 In vivo gene transfer of DNA encoding human growth hormone byretrograde injection of DNA into the salivary gland

A DNA expression construct encoding human growth hormone (hGH) isprepared by operably linking a CMV promoter to hGH-encoding DNA. Theexpression cassette is then inserted into a construct such as thebacterial plasmid pBR322. Escherichia coli is then transformed with theplasmid using conventional transformation procedures. E. coli containingthe plasmid are selected by virtue of the tetracycline or ampicillinresistance encoded by pBR322, and the transformed bacterial cellspropagated in culture. Plasmid DNA is then isolated from the transformedbacterial cell culture and the DNA purified by cesium gradient.

Approximately 250 μg of the purified plasmid DNA containing hGH DNA isinjected into the salivary gland of a human patient by retrograde ductalinjection via a salivary gland duct. Expression and intravenoussecretion of the protein is assessed using the method described above.

EXAMPLE 15 In vivo gene transfer of DNA encoding human growth hormone byretrograde ductal injection of naked DNA into the pancreas

A construct containing hGH-encoding DNA (Marshall et al., Biotechnology24:293-298, 1992) operably linked to the CMV promoter is resuspended in0.9% saline and a volume of the DNA solution is administered to a humanpatient. Approximately 1 mg of DNA is delivered to the pancreas of thepatient by cannulation of the pancreatic duct by duodenal intubationusing endoscopic retrograde cholangio-pancreatography. Expression andsecretion of human growth hormone into the bloodstream is assessed bydetection of the protein in the patient's blood.

EXAMPLE 16 In vivo gene transfer of DNA encoding human insulin bycannulation of naked DNA into the liver

A construct containing human insulin-encoding DNA operably linked to theCMV promoter is resuspended in 0.9% saline and a volume of the DNAsolution is administered to a human patient. Approximately 1 mg of DNAis delivered to the patient's liver by cannulation of the hepatic duct.Expression and secretion of human growth hormone into the bloodstream isassessed by detection of the protein in the patient's blood.

EXAMPLE 17 In vivo gene transfer of human insulin-encoding DNA to boththe pancreas and the liver of a patient

A construct containing human insulin-encoding DNA operably linked to theCMV promoter is resuspended in 0.9% saline. A volume of the DNA solutionis administered to a human patient so as to transform both pancreaticand hepatic cells (e.g., by introducing the DNA solution into the commonbile duct before it splits into the hepatic and pancreatic ducts).Approximately 2 mg of DNA is delivered to the patient's liver bycannulation of the hepatic duct; in addition, approximately 1 mg of DNAis delivered to the pancreas via retrograde injection via the pancreaticduct. Expression and secretion of human growth hormone into thebloodstream is assessed by detection of the protein in the patient'sblood.

Following procedures similar to those described above, other therapeuticproteins can be expressed from DNA inserted in the genome of a secretorygland cell by gene transfer according to the invention.

The invention now being fully described, it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade thereto without departing from the spirit or scope of the appendedclaims.

What is claimed is:
 1. A method of reducing blood glucose levels in adiabetic mammalian subject, the method comprising:introducing into thepancreas of the mammalian subject a construct comprising aninsulin-encoding DNA and a promoter sequence operably linked to theinsulin-encoding DNA to facilitate expression in a eukaryotic cell, saidintroducing being by intraductal administration; wherein expression ofthe introduced construct in the pancreas occurs such that a biologicallyactive insulin polypeptide is delivered into the bloodstream of thepatient in a therapeutically effective amount, and blood glucose levelsin the mammalian subject are reduced.
 2. The method of claim 1, whereina euglycemic state is maintained for at least three days.
 3. The methodof claim 1, wherein a euglycemic state is maintained for at least 6days.
 4. The method of claim 1, the method further comprising enhancingdelivery of the insulin polypeptide to the bloodstream by administrationof a cholinergic agonist to the mammalian subject.
 5. The method ofclaim 1, the method further comprising enhancing delivery of the insulinpolypeptide into the bloodstream by ingestion of a meal by the mammaliansubject.
 6. A method of delivering a desired polypeptide to a mammaliansubject, the method comprising:introducing in vivo into a lumen of aduct of a salivary gland and into a lumen of a duct of a liver of themammalian subject a construct comprising a DNA of interest that encodesthe desired polypeptide and a promoter sequence operably linked to theDNA of interest to facilitate expression in a eukaryotic cell; whereinthe introduced construct is expressed in each of the salivary gland andthe liver such that the polypeptide is delivered into the bloodstream ofthe mammal from each of the salivary gland and the liver.
 7. The methodof claim 6, wherein the DNA of interest encodes a factor VIIIpolypeptide.
 8. The method of claim 6, wherein the DNA of interestencodes a erythropoietin polypeptide.
 9. The method of claim 6, whereinintroduction into the salivary gland and into the liver is by retrogradeductal administration.
 10. The method of claim 6, wherein the constructintroduced into the salivary gland is not contained within a viralparticle.
 11. A method of delivering a polypeptide to a mammaliansubject, the method comprising:introducing in vivo into a lumen of aduct of a salivary gland and into a lumen of a duct of a pancreas of themammalian subject a construct comprising a DNA of interest that encodesa desired polypeptide and a promoter sequence operably linked to the DNAof interest to facilitate expression in a eukaryotic cell; wherein theintroduced construct is expressed in each of the salivary gland and thepancreas such that the polypeptide is delivered into the bloodstream ofthe mammalian subject from each of the salivary gland and the pancreas.12. The method of claim 11, wherein the desired polypeptide is selectedfrom the group consisting of insulin, growth hormone, interferon-alpha2b, Interferon-alpha 2a, interferon-alpha N1, filgastim, insulinotropin,imiglucerase, clotting factor VIII, interferon-beta1b, erythropoietin,sargramostim, interleukin-2, interferon-gamma, anti-CD3 antibody,GPIIb/IIIa monoclonal antibody, adenosine deaminase, interleukin-8,insulin-like growth factor-1, platelet-derived growth factor, epidermalgrowth factor, and hemoglobin.
 13. The method of claim 11, wherein theDNA of interest encodes a human insulin polypeptide.
 14. The method ofclaim 11, wherein the DNA of interest encodes a factor VIII polypeptide.15. The method of claim 11, wherein the DNA of interest encodes acrythropoietin polypeptide.
 16. The method of claim 11, whereinintroduction into the salivary gland and into the pancreas is byretrograde ductal administration.
 17. The method of claim 11, whereinthe construct introduced into the salivary gland is not contained withina viral particle.
 18. A method of delivering a polypeptide to amammalian subject, the method comprising:administering directly in vivointo a lumen of a duct of a pancreas and into a lumen of a duct of aliver of the mammalian subject a construct comprising a DNA of interestthat encodes a desired polypeptide and a promoter sequence operablylinked to the DNA of interest to facilitate expression in a eukaryoticcell; wherein the introduced construct is expressed in each of thepancreas and the liver such that the polypeptide is delivered into thebloodstream of the mammal from each of the pancreas and the liver. 19.The method of claim 18, wherein the desired polypeptide is selected fromthe group consisting of insulin, growth hormone, interferon-alpha 2b,Interferon-alpha 2a, interferon-alpha N1, filgastim, insulinotropin,imiglucerase, clotting factor VIII, interferon-beta1b, erythropoietin,sargramostim, interleukin-2, interferon-gamma, anti-CD3 antibody,GPIIb/IIIa monoclonal antibody, adenosine deaminase, interleukin-8,insulin-like growth factor-1, platelet-derived growth factor, epidermalgrowth factor, and hemoglobin.
 20. The method of claim 18, wherein theDNA of interest encodes a human insulin polypeptide.
 21. The method ofclaim 18, wherein the DNA of interest encodes a factor VIII polypeptide.22. The method of claim 18, wherein the DNA of interest encodes ancrythropoietin polypeptide.
 23. The method of claim 18, wherein saidadministering is by retrograde ductal administration.
 24. The method ofclaim 18, wherein said administering is by injection.
 25. The method ofclaim 18, wherein said administering is by cannulation.
 26. The methodof claim 25, wherein said cannulation is by inserting a cannula througha lumen of the gastrointestinal tract.
 27. The method of claim 25,wherein said cannulation is through an external orifice.
 28. The methodof claim 25, wherein said cannulation is by inserting a cannula througha common bile duct, wherein the DNA construct is delivered directly intothe hepatic duct and into the pancreatic duct.
 29. The method of claim18, wherein the introduced construct is expressed in each of thepancreas and the liver such that the level of polypeptide in pancreatictissue is elevated relative to a level of polypeptide in pancreatictissue after delivery of the same amount of the construct in the samemanner to pancreas alone.
 30. The method of claim 6, wherein the desiredpolypeptide is selected from the group consisting of insulin, growthhormone, interferon-alpha 2b, Interferon-alpha 2a, interferon-alpha N1,filgastim, insulinotropin, imiglucerase, clotting factor VIII,interferon-beta1b, erythropoietin, sargramostim, interleukin-2,interferon-gamma, anti-CD3 antibody, GPIIb/IIIa monoclonal antibody,adenosine deaminase, interleukin-8, insulin-like growth factor-1,platelet-derived growth factor, epidermal growth factor, and hemoglobin.31. The method of claim 6, wherein the DNA of interest encodes a humaninsulin polypeptide.